The goals of this research are to delineate cis-acting signals, to identify trans-acting viral and cellular factors, and to elucidate mechanisms that regulate cytomegalovirus (CMV) gene expression at the translational level. Investigations of the molecular basis of CMV translational control will reveal both CMV-specific translational processes, potentially useful as targets for antiviral interventions, as well as new insights into fundamentals of eukaryotic translation. This proposal focuses on translational regulation of the CMV glycoprotein gene gp48. The gp48 transcript leader contains an upstream open reading frame (uORF) which regulates downstream translation in transfection assays and which may account for the delayed expression of gp48 protein during CMV infection. Three specific goals will be pursued. (l) Investigations of naturally occurring gp48 leader variants and of mutants selected in vivo for loss of the uORF function will reveal the sequence requirements of the uORF inhibitory signal. (2) Analyses of gp48 protein synthesis during infection with wild-type and mutant strains of CMV will establish the role of the uORF signal, previously defined using transfection assays, in regulating expression of the authentic viral gene product. (3) Studies will evaluate and refine a model of gp48 translational regulation that predicts (i) that translation initiates at the AUG codon of the uORF infrequently, and (ii) that the inhibitory signal acts only in cis, even though (iii) the mediator of the inhibitory effect is the nascent peptide product of the uORF. Finally, development of a dependable cell-free translation assay will provide methods for investigating the mechanism underlying gp48 regulation and for identifying virally encoded or induced trans-acting factors. Although unusual among eukaryotic transcripts, uORFs are surprisingly common in transcripts of certain cellular genes including proto-oncogenes and growth factor genes. Thus, these studies of CMV gp48 will yield insights applicable to the control of cellular growth and differentiation as well as to the regulation of viral gene expression.